Filamentous fungal host cell strains have been engineered to express various proteins. These proteins can then be used, optionally after being purified, in various industrial, academic or other applications. The expression process can often be unpredictable. It is not a rare occasion when only a very small number, if any, of the transformants prepared actually produce the enzyme of interest. Variability in expression of heterologous genes (e.g., non-native genes, or native genes existing in a form that is different from the native form) can occur as a consequence of factors unrelated to their nucleic acid and/or amino acid sequences. For instance, non-homologous integration predominates in filamentous fungi. Thus, expression vectors integrate into the genome at random, possibly resulting in positional effects on expression levels between transformants. In addition, unstable transformants may be generated, necessitating further screening of transformants to obtain stable transformants. Variability may also occur by generation of heterokaryons as a result of transformation of a multinucleate protoplast. Therefore, reliable means of producing enzymes of interest, with reduced variability, provide clear advantages.
For certain industrial applications, these proteins produced from fungal host strains are often engineered to obtain new, desirable characteristics, or a different level of certain characteristics. In these cases, existing filamentous fungal host cell strains are often used for screening DNA libraries encoding variant proteins. Variability in expression efficacy and/or levels makes it difficult to compare the characteristics of a given variant with those of another. Therefore, a particular advantage is clearly present if variants can be reliably expressed if they can be expressed by the particular host cell, and if variants can be expressed at less variable levels such that their characteristics can be more readily assessed and compared.
While telomeric, extrachromosomal replicating vectors can be used as an alternative to genomic integration, this method does not eliminate variability in expression levels between transformants. Thus, the art would benefit from tools to reduce sequence-independent differences in gene expression from filamentous fungal host strains.